Cyclization-blocked proguanil as a strategy to improve the antimalarial activity of atovaquone

Abstract

Atovaquone-proguanil (Malarone®) is used for malaria prophylaxis and treatment. While the cytochrome bc1-inhibitor atovaquone has potent activity, proguanil’s action is attributed to its cyclization-metabolite, cycloguanil. Evidence suggests that proguanil has limited intrinsic activity, associated with mitochondrial-function. Here we demonstrate that proguanil, and cyclization-blocked analogue tBuPG, have potent, but slow-acting, in vitro anti-plasmodial activity. Activity is folate-metabolism and isoprenoid biosynthesis-independent. In yeast dihydroorotate dehydrogenase-expressing parasites, proguanil and tBuPG slow-action remains, while bc1-inhibitor activity switches from comparatively fast to slow-acting. Like proguanil, tBuPG has activity against P. berghei liver-stage parasites. Both analogues act synergistically with bc1-inhibitors against blood-stages in vitro, however cycloguanil antagonizes activity. Together, these data suggest that proguanil is a potent slow-acting anti-plasmodial agent, that bc1 is essential to parasite survival independent of dihydroorotate dehydrogenase-activity, that Malarone® is a triple-drug combination that includes antagonistic partners and that a cyclization-blocked proguanil may be a superior combination partner for bc1-inhibitors in vivo.

Novel Endochin-Like Quinolones Exhibit Potent In Vitro Activity against Plasmodium knowlesi but Do Not Synergize with Proguanil

“…As has been shown previously (10,13,23), atovaquone is synergistic in combination with proguanil against P. falciparum (Fig. 1A, Table 3).…”

“…Cycloguanil, like atovaquone, has been shown to be highly potent against P. knowlesi in vitro (20). Therefore, even though antagonistic interactions between atovaquone and cycloguanil have been described in vitro (13), the low nanomolar potency of both cycloguanil and atovaquone (20) should still support this combination for P. knowlesi infections, despite the reduced activity of proguanil, and its lack of synergy reported here.…”

“…ELQ-400 and ELQ-480 are both active at under 10 nM making them more potent than chloroquine but not as active as dihydroartemisinin (Table 1). P. falciparum exhibits significantly enhanced susceptibility to proguanil when incubated for more than one life cycle (13). Therefore, in preparation for in vitro combination analysis (isobolograms), we screened ELQ-300 and ELQ-400 as well as proguanil and atovaquone using a longer incubation time (2.5 life cycles).…”

“…Based on these data, combination studies were then designed to explore the in vitro interactions between the compounds. These experiments were also run over multiple life cycles to take into account the increased potency of proguanil after longer exposures (13).…”

“…Atovaquone is therefore used in combination with a synergistic partner drug, proguanil (10), but even this combination is vulnerable to mutations in pfcytb especially in areas of cycloguanil resistance (11,12). Although the target of proguanil is not currently known, proguanil has recently been shown to increase in potency against Plasmodium falciparum after a longer in vitro exposure (13). Considering that the mitochondrial targeting atovaquone-proguanil combination differs in its mechanism of resistance to the components of current ACT, this combination has been investigated as an alternative treatment of multidrug resistant malaria infections (14), though it should be noted that the atovaquone-proguanil combinations tested performed poorly (only 90-92% effective at 42 days post-treatment) in that region of Cambodia, and are unlikely to be useful as treatment.…”